Evidence for histidine in the active site of papain.
Papain was irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. The molecular weight of the inhibited enzyme was indistinguishable from that of papain itself, and no evidence of dimeric or oligomeric species was found. The optical-rotatory-dispersion curves of chloroacetone-inhibited papain and 1,3-dibromoacetone-inhibited papain were essentially similar. Amino acid analysis of the 1,3-dibromo[2-(14)C]acetone-inhibited enzyme and the performic acid-oxidized material clearly showed that a cysteine and histidine residue had been alkylated through the thiol and N-1 of the imidazole group respectively. These groups must therefore be within 5å of each other in the tertiary structure of papain. Possible mechanistic implications are briefly discussed.
D000096:Acetone; D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001966:Bromine; D002247:Carbon Isotopes; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D003545:Cysteine; D006639:Histidine; D007093:Imidazoles; D008956:Models, Chemical; D008970:Molecular Weight; D009905:Optical Rotatory Dispersion; D010206:Papain / Q000032:analysis / Q000037:antagonists & inhibitors; D010944:Plants / Q000201:enzymology*; D013438:Sulfhydryl Compounds
D000596:Amino Acids; D002247:Carbon Isotopes; D007093:Imidazoles; D013438:Sulfhydryl Compounds; D000096:Acetone; D006639:Histidine; D010206:Papain; D003545:Cysteine; D001966:Bromine